The ability to cross the cell membrane and gain access to the cell interior is a requirement for any therapeutic agent intended for an intracellula target. Proteins, peptides, and small interfering RNA (siRNA) represent some of the emerging/expanding classes of therapeutic agents. However, these biological molecules are generally membrane impermeable on their own and must be delivered to the proper intracellular locations. Cell-penetrating peptides (CPPs) are short peptides (usually <30 amino acids) that have the ability to penetrate the plasma membrane of eukaryotic cells without causing significant membrane damage. Many CPPs have been discovered and some have been used to deliver a wide variety of cargos into cells. A major limitation of previous CPPs is that they have poor overall delivery efficiency due to inefficient endosomal escape. A new family of cyclic CPPs, e.g., cyclo(F?RRRRQ) (where ? is 2-naphthylalanine), was recently discovered in this laboratory and found to escape from the endosome efficiently. The overall objective of this project is to elucidate the mechanisms of CPP uptake and endosomal escape and develop more potent CPPs as general transporters for intracellular delivery of biological active molecules. Specific Aim 1 is to synthesize and screen a focused cyclic peptide library to identify additional and potentially more active cyclic CPPs. Specific Aim 2 is to test the limits of what ca be delivered by the cyclic CPPs and determine the mechanism of CPP endosomal release as well as the factors that control the endosomal escape efficiency. Finally, Specific Aim 3 is to utilize the cyclic CPPs to design cell permeable inhibitors against intracellular proteins such as the oncoprotein Ras.